We shall complete a detailed characterization of the structure and replication requirements of specific reiteration mutants of simian virus 40. These mutants have tandem repeats of small segments of the SV40 genome including the initiation site for DNA replication. Using restriction endonucleases, gel electrophoresis and electron microscopy we will compare the host DNA substitutions in these mutants to each other and to possible similar sequences surrounding integrated viral genomes in transformed cells. Since these mutants possess multiple copies of the origin for replication, we shall employ them to detect initiation of replication in vitro. The gene A protein of SV40 will subsequently be purified with a complementation assay based on its involvement in the initiation of DNA replication. These studies of reiteration mutants have led us into a series of experiments on the fate of small segments of phage lambda DNA in monkey cells. In this recombinant DNA work, we covalently joined two segments from the immunity region of phage lambda to SV40 (vector) DNA segments isolated from two different reiterations. More recently we have constructed and cloned a SV40 hybrid carrying a bacterial nonsense suppressor tRNA gene in the late gene region of SV40. We shall pursue studies of the transcription and stability of these prokaryotic sequences both in permissive cells and, in the case of the suppressor hybrid, in rat cells transformed by this hybrid DNA.